Distribution of nerve growth factor in the submandibular gland of the male and female mouse

Summary Nerve growth factor (NGF) was localized in the mouse submandibular gland by means of indirect immunofluorescence applied to 0.5 mthick sections of freeze-dried, plastic-embedded tissue. The antibody to NGF (I_gG-fraction) was raised in rabbits immunized with pure 2.5 S NGF from submandibular glands of adult male mice.In the male gland anti-NGF bound selectively to the secretory granules was present in the cells of the granular ducts. Immunoreactive granules extended from the perinuclear region toward the apical pole. In the female gland immunoreactive cells and granules were considerably less abundant than in males. Immunofluorescence was confined to individual secretory cells located in the wall of the granular striated duct.In the present study no support was found for the hypothesis suggesting that immunoreactive NGF is formed within the secretory granules during their transport from the perinuclear region to the apical pole.     

Cytochemical detection of endogenous peroxidase in the acinar cells of the hamster submandibular gland

The presence of endogenous peroxidase activity in the hamster submandibular gland was investigated cytochemically by light and electron microscopy using diaminobenzidine methods. After fixation of tissue with 2% paraformaldehyde--2.5% glutaraldehyde and incubation in a DAB reaction medium containing 0.01% H₂O₂, the peroxidase reaction product was localized in the nuclear envelope, the cisternae of the endoplasmic reticulum, secretory granules and the Golgi apparatus in both the acinar and granular duct cells of the submandibular gland. This is in contrast to earlier investigators who failed to detect peroxidase activity in acinar cells of the hamster submandibular gland and reported that peroxidase is localized only in the granular duct cells. The discrepancy may be caused by differences in experimental procedures. It is suggested that fixation of tissue with a high concentration of glutaral dehyde and incubation in a DAB reaction medium containing a high concentration of H₂O₂ inhibits the peroxidase activity of acinar cells in the hamster submandibular gland     

Cytochemical detection of endogenous peroxidase in the acinar cells of the hamster submandibular gland

The presence of endogenous peroxidase activity in the hamster submandibular gland was investigated cytochemically by light and electron microscopy using diaminobenzidine methods. After fixation of tissue with 2% paraformaldehyde--2.5% glutaraldehyde and incubation in a DAB reaction medium containing 0.01% H₂O₂, the peroxidase reaction product was localized in the nuclear envelope, the cisternae of the endoplasmic reticulum, secretory granules and the Golgi apparatus in both the acinar and granular duct cells of the submandibular gland. This is in contrast to earlier investigators who failed to detect peroxidase activity in acinar cells of the hamster submandibular gland and reported that peroxidase is localized only in the granular duct cells. The discrepancy may be caused by differences in experimental procedures. It is suggested that fixation of tissue with a high concentration of glutaral dehyde and incubation in a DAB reaction medium containing a high concentration of H₂O₂ inhibits the peroxidase activity of acinar cells in the hamster submandibular gland This revised version was published online in November 2006 with corrections to the Cover Date.     

Immunocytochemical demonstration of nerve growth factor and histofluorescence of catecholaminergic nerves in the salivary glands of diabetic mice

Summary Nerve growth factor (NGF) was localized in the submandibular, sublingual, and parotid salivary glands of male and female diabetic mice and their normal littermates by immunoperoxidase staining usingp-phenylenediamine-pyrocatechol as a chromogen for the cytochemical demonstration of peroxidase activity. In the normal male submandibular gland, immunoreactive NGF was localized in the apical regions of granular, intercalated and collecting duct cells, while in the normal female submandibular gland, NGF was present throughout the cytoplasm of granular duct cells. The localization of NGF in the diabetic male and female submandibular glands was similar and resembled that of the normal female. NGF immunoreactivity was also observed in the striated duct cells in the sublingual and parotid glands of all four types of mice.The sympathetic innervation of the submandibular glands of normal and diabetic mice was demonstrated using glyoxylic acid-induced histofluorescence. The pattern of sympathetic innervation and the intensity of catecholamine fluorescence was consistently different in the four types of mice. In the normal male submandibular gland the fluorescence was very intense, particularly in nerves adjacent to the granular ducts. In the normal female submandibular gland, the fluorescence was weak, while in the diabetic male and female the fluorescence was moderate.The correlation between the intensity of the immunocytochemical staining for NGF and the catecholamine fluorescence adjacent to the granular ducts suggests a trophic influence of the NGF-containing granular ducts on their sympathetic innervation.     

Basic fibroblast growth factor in rat salivary glands

We studied the occurrence and localization of basic fibroblast growth factor (bFGF) in rat salivary glands using a specific monoclonal antibody. It was shown that the extract of rat salivary glands has a pronounced stimulatory activity on the growth of bovine capillary endothelial cells, which is blocked by the addition of an antibody against bFGF. The concentration of bFGF in the submandibular/sublingual gland, as determined by radioimmunoassay, was 80% that in the brain. Immunocytochemistry revealed bFGF-immunoreactivity localized primarily in the epithelial cells lining the striated ducts and excretory ducts of the parotid, sublingual and submandibular glands. In addition, intense bFGF-immunoreactivity was observed in the granular convoluted tubule of the submandibular gland, localized predominantly in the agranular pillar cells, which lay in small numbers among the majority of weakly immunostained cells containing many apical secretory granules. At the electron-microscopic level, the immunoreactive material was distributed diffusely in the cytoplasmic matrix and nuclei of all immunoreactive cells, whereas it was absent from all cytoplasmic organelles including the secretory granules. These results indicate that bFGF is localized in different cellular and subcellular compartments from those of other growth factors in the duct system of rat salivary glands.     

Fine structure of submandibular glands of mice with testicular feminization (Tfm/Y)

Summary The fine structure of the submandibular gland of the mouse with testicular feminization (Tfm/Y) was studied by light and electron microscopy. The architecture of the Tfm/Y gland proved to be rather similar to that of the normal female mouse in both tubular ratio and structure. Granular convoluted tubular cells in Tfm/Y mice characteristically had fewer secretory granules and increased cytoplasmic vacuoles than normal littermates, suggesting an altered synthesis of secretory granules in this cell type of the Tfm/Y mouse. Moreover, there were differences in the ultrastructure of submandibular glands between Tfm/Y and normal female mice. In the gland of the Tfm/Y mouse, basal striations of the striated secretory tubular cells were not so developed and granular intercalated duct cells were less than those of normal females. These findings support the evidence that the secretory tubule of the mouse submandibular gland responds to androgens, resulting in accentuated development in the male, while also suggesting the possibility that the mouse submandibular gland is regulated by other factors which lead to the prominent sexual dimorphism observed in this gland.     

An ultrastructural study of the prostate gland of megascolicid earthworm Lampito mauritii (Kinberg)

The ultrastructure of prostate gland of Lampito mauritii revealed two types of secretory cells. Type 1 cells with a broad basal region and a long apical region contain electron dense oval secretory granules with an increased density at the core region. Numerous electron lucent granules with fine filamentous and electron dense amorphous materials also occur at the basal region of these cells. Type 2 cells contain electron lucent mucous-like secretory granules. This cell type contains exceptionally large Golgi complexes having 20-23 stacked cisternae. Both cell types open into a common lumen and numerous microtubules are visible at the apical end. Junctional complexes, such as desmosomes and septate junctions, are observed in this glandular tissue.     

Immunocytochemical localization of renin in the submandibular gland of the mouse.

Renin was localized in the submandibular gland of the adult mouse at light and electron microscopic levels by the unlabeled antibody enzyme method of Sternberger. At the light microscopic level, renin was confined to the granular convoluted tubule (GCT) segment of the gland with considerable variation among GCT cells in intensity of staining. Some GCT cells failed to stain for renin. The pattern of staining was the same in the gland of male and female mice, but in the glands of females GCT segments were smaller and less numerous. At the electron microscopic level, staining for renin was also confined to the GCT cells, and was localized exclusively to the secretory granules. The intensity of staining of the secretory granules within a given GCT cell varied; some cells contained only minimally reactive or negative secretory granules. All other organelles within the GCT cell, except condensing vacuoles, failed to stain.     

Cytodifferentiation of striated duct cells and secretory cells of the convoluted granular tubules of the rat submandibular gland.

The structural and functional development of the striated ducts and convoluted granular tubules (CGT) of the rat submandibular gland (SMG) were studied by electron microscopy and alkaline protease chemistry. Development of the SMG was followed from 14 days of gestation through 30 weeks of age. The specialized morphology of the basal aspect of the striated duct cells arises from cellular extensions which are first seen at 20 days of gestation. These processes elongate and intertwine with similar processes from adjacent cells, and as the cells enlarge the processes are compressed together giving the appearance of "infolding" of the basal plasma membrane. Mitochondria migrate to the basal part of the cell and are seen in close relationship to the cellular extensions throughout the development of these cells. Development of the striated duct is complete by one week after birth. The CGT develop from the proximal portions of intralobular striated ducts. At one week after birth, cells of the proximal striated duct demonstrate apical vacuoles. By two weeks after birth these vacuoles are replaced by distinct zymogen-like granules. There is a progressive accumulation of large numbers of secretory granules in the CGT cells as the animals age. However, rough endoplasmic reticulum is a relatively inconspicuous cellular component throughout development. The accumulation of alkaline protease activity in the gland closely parallels the pattern of granule accumulation.     

Histology and histochemistry of the parotid and the principal and accessory submandibular glands of the little brown bat

The parotid and the principal and accessory submandibular glands of the little brown bat. Myotis lucifugus (Vespertilionidae), were examined using light microscopy and staining methods for mucosubstances. The parotid gland is a compound tubuloacinar seromucous gland. Parotid gland secretory cells contain both neutral and nonsulfated acidic mucosubstances. The principal and accessory submandibular glands are compound tubuloacinar mucus-secreting glands. They contain somewhat atypical mucus-secreting demilunar cells that often appear to be interspersed between mucous tubule cells. The mucous tubule cells in both the principal and accessory submandibular glands contain sulfonmucins. Demilunar cells of the principal submandibular gland contain moderate amounts of nonsulfated acidic mucosubstances, but the corresponding cells of the accessory submandibular gland contain considerable neutral mucosubstance with very little acid mucosubstance. Intercalated ducts composed of cuboidal or low columnar epithelial cells are present in all three glands. Striated ducts in all glands are composed of columnar cells whose apices bulge into the ductal lumina. Excretory ducts are composed of simple columnar epithelium, with occasional basal cells that suggest a possible pseudostratified nature. The cells of the excretory ducts also have bulging apices. All duct types contain apical cytoplasmic secretory material that is a periodic acid-Schiff positive, neutral mucosubstance. Ductal apical secretory material is more evident in intercalated and striated ducts than in excretory ducts.     

The influence of fixation on the carbohydrate cytochemistry of rat salivary gland secretory granules

Synopsis A series of studies was performed to assess the optimum fixation conditions for staining of carbohydrate-containing constituents of rat salivary gland secretory granules. In the parotid and submandibular salivary glands of the rat, the reactivity of secretory granules, at both the light and electron microscopic level, with routine stains and with cytochemical reagents was highly dependent upon the nature of the fixative employed. At the light microscopic level, secretory granules in rat parotid gland were periodic acid-Schiff (PAS) positive if fixed with buffered formalin fixatives. However, if the gland was fixed with lipid-solvent-containing fixatives, or with formalin at a very acid pH (as in Bouin's fixative), the PAS reactivity of the granules was lost. In the submandibular gland of rats, the acinar cells and granular tubules behaved similarly after such fixation in terms of their PAS reactivity, particularly in males; acinar cells of the female submandibular gland stained only lightly with PAS. At the fine structural level, the morphology of secretory granule constituents depended on the buffer used (cacodylate, phosphate or collidine) and on whether or not tissue was post-osmicated. Post-osmication considerably reduced the reaction of secretory granule components with stains for carbohydrates.The experimental evidence indicated that the carbohydrate-containing components of both parotid and submandibular gland secretory granules were not typical long-chain neutral or acidic mucins, but were rather glycolipids or lipophilic glycoproteins that were solubilized by lipid solvents or at acidic pH and were lost or destroyed in the presence of strong oxidants.     

Rhipicephalus (Boophilus) microplus (Canestrini, 1887) (Acari: Ixodidae): acid phosphatase and ATPase activities localization in salivary glands of females during the feeding period

This study investigates the presence and the localization of acid phosphatase and ATPase in the salivary glands of Rhipicephalus (Boophilus) microplus female ticks during feeding. Semi-engorged females showed a larger amount of acid phosphatase compared to those at beginning of feeding, localized mainly in the apical portion of the secretory cells, and in the basal labyrinth of the interstitial cells. Ultrastructural observations also demonstrated its presence in secretion granules and inside some nuclei of secretory cells at beginning of feeding. Acid phosphatase in a free form probably has a hemolymph and/or ribosomal origin and participates in salivary gland secretion control. ATPase was detected in basal membrane of all types of acini and/or in the cytoplasm of the secretory cells at both feeding stages. The enzyme activities found strongly suggests that cell death by apoptosis occurs during the degenerative process.     

Cytochemical studies of GERL and its role in secretory granule formation in exocrine cells

Synopsis the structure and cytochemistry of GERL was studied in several different exocrine secretory cells, including the exorbital lacrimal gland, parotid, lingual serous (von Ebner's), submandibular, and sublingual salivary glands, and exocrine pancreas of the rat; the lacrimal, parotid and pancreas of the guinea-pig; and the lacrimal gland of the monkey. GERL was morphologically and cytochemically similar in all cell types studied. It was located in the inner Golgi region and consisted of cisternal and tubular portions. Immature secretory granules were in continuity with GERL through multiple tubular connections. Modified cisternae of endoplasmic reticulum, with ribosomes only on one surface, closely paralleled parts of GERL. GERL and immature granules were intensely reactive for acid phosphatase activity, while the inner Golgi saccules were reactive for thiamine pyrophosphatase and nucleoside diphosphatase activities. In the rat exorbital lacrimal and parotid glands, reaction product for endogenous peroxidase, a secretory enzyme, was present in the endoplasmic reticulum, Golgi saccules, immature and mature secretory granules. GERL was usually free of reaction product or contained only a small amount. The widespread occurrence of GERL in secretory cells, and its intimate involvement with the formation of granules, suggest that it is an integral component of the secretory process.     

Immunocytochemical localization of nerve growth factor: effects of fixation

The fixation dependence of immunocytochemically demonstrable nerve growth factor (NGF) was investigated. Several commonly used fixation methods have been employed, including buffered formaldehyde, Bouin's fluid, and chloroform-methanol, as well as freezing and cryostat sectioning. The immunostaining technique was an immunoenzyme bridge procedure on either paraffin sections or frozen sections. Of those methods tested, fixation for 1 hr in a buffered formaldehyde appeared to provide optimal preservation and localization of immunoreactive material. Using this method, reaction product was localized in granules of the granular tubule cells of the male mouse submandibular gland. Prolonged fixation in buffered formaldehyde resulted in a diffuse background staining and loss of granule immunoreactivity. In frozen sections and in tissues fixed with either Bouin's solution, chloroform-methanol, or buffered paraformaldehyde-glutaraldehyde increased cytoplasmic background staining and loss of granule immunoreactivity were observed. It was concluded that, for the localization of NGF at the light microscopic level, a brief (1 hr) buffered formaldehyde fixation is optimal.     

Morphology and ultrastructure of the venom glands of the northern pacific rattlesnake Crotalus viridis oreganus

The venom gland of Crotalus viridis oreganus is composed of two discrete secretory regions: a small anterior portion, the accessory gland, and a much larger main gland. These two glands are joined by a short primary duct consisting of simple columnar secretory cells and basal horizontal cells. The main gland has at least four morphologically distinct cell types: secretory cells, the dominant cell of the gland, mitochondria-rich cells, horizontal cells, and “dark” cells. Scanning electron microscopy shows that the mitochondria-rich cells are recessed into pits of varying depth; these cells do not secrete. Horizontal cells may serve as secretory stem cells, and “dark” cells may be myoepithelial cells. The accessory gland contains at least six distinct cell types: mucosecretory cells with large mucous granules, mitochondria-rich cells with apical vesicles, mitochondria-rich cells with electron-dense secretory granules, mitochondria-rich cells with numerous cilia, horizontal cells, and “dark” cells. Mitochondria-rich cells with apical vesicles or cilia cover much of the apical surface of mucosecretory cells and these three cell types are found in the anterior distal tubules of the accessory gland. The posterior regions of the accessory gland lack mucosecretory cells and do not appear to secrete. Ciliated cells have not been noted previously in snake venom glands. Release of secretory products (venom) into the lumen of the main gland is by exocytosis of granules and by release of intact membrane-bound vesicles. Following venom extraction, main gland secretory and mitochondria-rich cells increase in height, and protein synthesis (as suggested by rough endoplasmic reticulum proliferation) increases dramatically. No new cell types or alterations in morphology were noted among glands taken from either adult or juvenile snakes, even though the venom of each is quite distinct. In general, the glands of C. v. oreganus share structural similarities with those of crotalids and viperids previously described.     

Kallikrein localization in rodent salivary glands and kidney with the immunoglobulin-enzyme bridge technique.

Kallikrein has been localized in rodent kidney and salivary glands by means of an immunoglobulin-enzyme bridge technique. In sections of kidney, anti-kallikrein antibodies bound to the apical region of certain distal tubule segments in the cortex, to reabsorption droplets of proximal convoluted tubules, and to certain duct segments in the papilla. In salivary glands of both male and female rats and mice, and apical rim of most striated duct cells of submandibular, parotid and sublingual glands and granular tubules of submandibular glands exhibited immunoreactivity. Granular intercalated duct cells in female submandibular glands also displayed immunostaining for kallikrein. Phenylephrine administration resulted in loss of immunoreactive granules from the granular convoluted tubule cells of male mouse submandibular gland. This response was paralleled by a biochemically demonstrable decrease in kallikrein-like tosylarginine methyl ester (TAME) esterase activity.     

Immunocytochemical localization of nerve growth factor in mouse salivary glands

Summary The submandibular glands of female mice and the sublingual and parotid glands of adult male and female mice have been examined by light microscopical immunocytochemistry for nerve growth factor (NGF). In female submandibular glands, staining for NGF was observed in granular convoluted tubule and striated duct cells. Sublingual glands of the mouse contained relatively few granular cells staining for NGF compared with submandibular glands. However, such granular cells appeared to be more numerous in male sublingual glands than in female glands. The remainder of the intralobular duct cells in both male and female sublingual glands exhibited apical subluminal staining for NGF as well as light basal plasmalemmal staining. Parotid glands in both male and female mice exhibited a similar pattern of staining for NGF in striated duct cells. However, the glands did not contain granular cells nor did they exhibit any pattern of staining which reflected a sexual dimorphism. Immunodot staining of salivary gland extracts confirmed the presence of immunoreactivity for NGF in all three of the major salivary glands.     

Fine structure of the mandibular gland of the Djungarian hamster (Phodopus sungarus)

The mandibular gland of the Djungarian hamster was examined by light microscopy, and transmission and scanning electron microscopies. Its acinar cells reacted with periodic acid-Schiff (PAS) and were weakly stained with alcian blue (AB). There were intercellular canaliculi between the acinar cells. These cells therefore appeared to be seromucous. The acinar epithelium was composed of light cells containing various spherical secretory granules. The granular cells of the mandibular gland possessed many acidophilic granules exhibiting a positive reaction to PAS stain. They were frequently observed at the junction of the acini and intercalated ducts in all mandibular glands examined. All of these cells were light and contained secretory granules of varying size and density. The intercalated ducts consisted exclusively of light cells possessing a few round granules of high density in the apical region. The striated ducts were comprised of two portions--a secretory portion and a typical striated portion without secretory granules. The secretory portion consisted of light, dark and specifically light epithelial cells containing acidophilic granules, which exhibited a strongly positive PAS reaction. The epithelium of typically striated portions was composed of light and dark cells containing fine vacuoles in the apical region. The mandibular gland of the Djungarian hamster revealed no histological differences between sexes.     

Ultrastructure and complex carbohydrate ultracytochemistry of the cat parotid gland

The structure and glycoconjugate content of the cat parotid gland were analyzed at electron microscopic level by applying morphological techniques and three ultrastructural histochemical methods - HID-TCH-SP, LID-TCH-SP and PA-TCH-SP. This gland appeared as a typical salivary gland composed of acinar secretory cells, intercalated ducts, striated ducts and excretory ducts. The most common configuration of secretory granules consisted of a dense core surrounded by a variable electron-lucent halo. All ductal segments were characterized by the presence of different cell populations and small apical granules greatly different from those localized in the acinar cells. By using HID-TCH-SP we were able to demonstrate that in a few acinar cells there are sulphated sites, whereas PA-TCH-SP staining revealed the presence of vic-glycol radicals in all acinar cells preferentially located on the halo of secretory granules.     

Acid phosphatase and peroxidase in ‘resting’ acinar cells of the major salivary glands of cats and their possible movement into secretory granules

Synopsis After fixation by perarterial perfusion using an aldehyde mixture, salivary tissues were prepared for ultrastructural cytochemistry of acid phosphatase or peroxidase. Great variations in the distributions of the reaction products occurred, often within the same cell. Acid phosphatase staining occurred not only in lysosomes and sometimes in a GERL system, but a diffuse cytoplasmic component was also found in submandibular central acinar cells and to a lesser extent in parotid acini and variable staining occurred in the secretory granules of these cells. Peroxidase was variably associated with rough endoplasmic reticulum in submandibular demilunar cells, parotid acini, and more strongly in some sublingual cells. The secretory granules of the latter were darkly stained, but in parotid granules there was varibale staining and least staining occurred in the granules of submandibular demilunes.These results are thought to indicate that not all enzymes present in secretory granules have reached there by an elective secretory process. Sometimes they appear to have entered the granules haphazardly, possibly having been enzymes associated with intracellular cisternal channels for transport or metabolism of other secretory substances and ultimately to have passed into the cisternal channels by chance or as part of a natural removal of redundant material.